Bale, Laurie K.

Pregnancy-associated plasma protein A (PAPPA) is a metalloproteinase that controls the tissue availability of insulin-like growth factor (IGF). Homozygous deletion of PAPPA in mice leads to lifespan extension. Since immune function is an important determinant of individual fitness, we examined the natural immune ecology of PAPPA −/− mice and their wild-type littermates reared under specific pathogen-free condition with aging. Whereas wild-type mice exhibit classic age-dependent thymic atrophy, 18-month-old PAPPA −/− mice maintain discrete thymic cortex and medulla densely populated by CD4 + CD8 + thymocytes that are capable of differentiating into single-positive CD4 and CD8 T cells. Old PAPPA −/− mice have high levels of T cell receptor excision circles, and have bone marrows enriched for subsets of thymus-seeding progenitors. PAPPA −/− mice have an overall larger pool of naive T cells, and also exhibit an age-dependent accumulation of CD44 + CD43 + memory T cells similar to wild-type mice. However, CD43 + T cell subsets of old PAPPA −/− mice have significantly lower prevalence of 1B11 and S7, glycosylation isoforms known to inhibit T cell activation with normal aging. In bioassays of cell activation, splenic T cells of old PAPPA −/− mice have high levels of activation antigens and cytokine production, and also elicit Ig production by autologous B cells at levels equivalent to young wild-type mice. These data suggest an IGF-immune axis of healthy longevity. Controlling the availability of IGF in the thymus by targeted manipulation of PAPPA could be a way to maintain immune homeostasis during postnatal development and aging.

Abstract IGFBP-4 is an inhibitor of IGF-I in bone. We show that TGF-β regulates IGFBP-4 and enhances IGF-I-stimulated growth of cultured human bone cells through increased expression of an IGFBP-4 protease, PAPP-A. This effect of TGF-β on IGF-I bioavailability may promote local bone formation. Insulin-like growth factor binding protein (IGFBP-4) proteolysis is implicated in the regulation of local insulin-like growth factor (IGF)-I bioavailability during bone remodeling. The IGFBP-4 protease secreted by normal adult human osteoblastic (hOB) cells in culture is a novel metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). We have recently identified an inhibitor of PAPP-A, the precursor form of major basic protein (proMBP). Very little is known about the molecular regulation of this IGFBP-4 protease system. In the present study, we determined the effect of transforming growth factor (TGF)-β and IGF-II, the two most abundant growth factors in human bone, on PAPP-A and proMBP expression in primary cultures of hOB cells. Treatment with TGF-β resulted in time- and dose-dependent increases in PAPP-A mRNA expression, with a maximal 12-fold increase after 24 h of stimulation with 10 ng/ml TGF-β. Increased PAPP-A levels in hOB cell-conditioned medium paralleled PAPP-A gene expression. In addition, TGF-β completely suppressed proMBP expression. Treatment of hOB cells with IGF-II had no effect on PAPP-A or proMBP gene expression. However, IGFBP-4 proteolysis in cell-free assay was dependent on IGF-II, and there was increased IGF-II-dependent IGFBP-4 protease activity in conditioned medium from hOB cells that were treated with TGF-β. IGF-I stimulation of hOB cell proliferation was markedly enhanced by pretreatment with TGF-β and [Leu27]IGF-II, and this enhancement was prevented with protease-resistant IGFBP-4. In summary, TGF-β regulates IGFBP-4 proteolysis in hOB cells through increased expression of the protease, PAPP-A, and decreased expression of the inhibitor, proMBP. However, functional activation of the IGFBP-4 protease system is dependent on IGF-II, which acts at a post-translational level. These data support a model whereby local TGF-β and IGF-II in the bone microenvironment coordinately amplify IGF-I bioavailability through controlled IGFBP-4 proteolysis, which may be a means to promote bone formation.

Pregnancy-associated plasma protein A (PAPPA) is a metzincin superfamily metalloproteinase in the insulin-like growth factor (IGF) system. PAPPA increases IGF bioavailability and mitogenic effectiveness in vitro through regulated cleavage of IGF-binding protein 4 (IGFBP4). To determine its function in vivo, we generated PAPPA-null mice by gene targeting. Mice homozygous for targeted disruption of the PAPPA gene were viable but 60% the size of wild-type littermates at birth. The impact of the mutation was exerted during the early embryonic period prior to organogenesis, resulting in proportional dwarfism. PAPPA, IGF2 and IGFBP4 transcripts co-localized in wild-type embryos, and expression of IGF2 and IGFBP4 mRNA was not altered in PAPPA-deficient embryos. However,IGFBP4 proteolytic activity was completely lacking in fibroblasts derived from PAPPA-deficient embryos, and IGFBP4 effectively inhibited IGF-stimulated mitogenesis in these cells. These results provide the first direct evidence that PAPPA is an essential growth regulatory factor in vivo, and suggest a novel mechanism for regulated IGF bioavailability during early fetal development.

AbstractThe insulin receptor mediates a proliferative response in certain transformed cells, but little is known about its function in ovarian cancer. We used human epithelial ovarian carcinoma cell lines and lifespan-extended normal ovarian surface epithelial (OSE) cells to examine 125I-insulin binding and mitogenic responses to insulin. All cancer cell and OSE cultures specifically bound 125I-insulin. Except for OV202, the carcinoma lines had elevated insulin binding compared with OSE cells. All carcinoma lines except OV202 expressed insulin receptor as detected by flow cytometry and increased 3H-thymidine incorporation or cell number in response to 0.1–10 nm insulin. Interestingly, similar concentrations of IGF-II also induced proliferation of the insulin-responsive cancer cell lines and displaced 125I-insulin binding. Direct binding of 125I-IGF-II to the insulin receptor was visualized by cross-linking and immunoprecipitation. Binding of IGF-II to the insulin receptor and a proliferative effect of insulin suggest the presence of insulin receptor isoform A. Real-time PCR analyses confirm that insulin receptor isoform A expression predominates over isoform B expression in the ovarian carcinoma cell lines. This report suggests that the insulin receptor may play a role in the regulation of ovarian cancer cell growth.